An attempt to conjugate a drug to a water-soluble polymer has been generally made for the purposes to improve properties such as prolonged residence time in blood, improved stability and solubility and reduced antigenicity of a low-molecular-weight drug, a peptide drug, a protein drug, or the like. In particular, polyethylene glycol (hereinafter also referred to as “PEG”) has been widely used because of its inert properties and also because it has the effect of preventing adsorption of a protein to a drug in vivo. A PEG-conjugated protein has already been at the practical stage as a pharmaceutical. However, PEG is not a biodegradable polymer. Therefore, when a PEG-conjugated protein is accumulated in a body as a result of a long-term administration, problems regarding safety and the like have not yet been solved. Moreover, a phenomenon whereby the clearance of the 2nd administration is abnormally accelerated in a PEG-conjugated liposome (Accelerated Blood Clearance phenomenon; hereinafter also referred to as “ABC phenomenon”) has recently been reported (refer to Non-Patent Documents 1 and 2). Thus, it is hard to say that the safety and effectiveness of such a PEG-conjugated pharmaceutical has sufficiently been established.
Hyaluronic acid (hereinafter also referred to as “HA”) is a polysaccharide isolated from the vitreous body of the bovine eye by K. Meyer in 1934. It has been well known as a main component of an extracellular matrix for a long time. HA is one type of glycosaminoglycan consisting of disaccharide units formed by connecting D-glucuronic acid to N-acetylglucosamine via a β (1→3) glycoside bond. Hyaluronic acid does not have species difference regarding the chemical and physical structure thereof, and a hyaluronic acid metabolic system exists also in humans. Furthermore, it can also be said that hyaluronic acid is an extremely safe biomaterial in terms of immunity and toxicity. In recent years, the aspect of hyaluronic acid as a physiologically active substance associated with cell adhesion, cell growth, and induction of cell migration has been focused. Further, as a result of the mass production of high-molecular-weight hyaluronic acid that has been succeeded using microorganisms, hyaluronic acid has been in the practical use as a pharmaceutical such as a therapeutic agent for articular disease. Still further, hyaluronic acid has also been practically used in the field of cosmetics, etc. Still further, it has been reported that conjugation of a drug to hyaluronic acid enables targeting of the drug to cancer tissues (refer to Patent Document 1), targeting to the liver (refer to Patent Document 2), a reduction in antigenicity (refer to Patent Document 3), elongation of the residence time in blood (refer to Patent Documents 4, 5, and 6), etc.
Compared with a generally used PEG, the use of hyaluronic acid as a drug conjugate carrier is advantageous in that HA has biodegradability, in that an enormous size of HA is available, and also in that since HA has many reactive sites in a molecule thereof, multiple drugs (a plurality of single drugs, or two or more drugs) can be linked in a single molecule. A use of hyaluronic acid having such advantages as a drug conjugate carrier becomes means for designing and developing a conjugate having higher functions to control drug pharmacokinetics, such as targeting or sustained-release. In addition, since hyaluronic acid is biodegradable and does not have species difference in terms of the chemical structure thereof, it can be said that such hyaluronic acid is a carrier that is more excellent than PEG from the viewpoint of safety as well.
However, it has been reported that the residence time of hyaluronic acid itself in blood is short, and that the half-life period thereof is 2 minutes after an intravenous injection (hereinafter also referred to as “iv”) (refer to Non-Patent Document 3). Also in the studies by the present inventors, when hyaluronic acid was just conjugated to a drug, a significant elongation of the residence time of the drug in blood or the improvement of the persistence of the drug effect was not confirmed. The main metabolic sites of hyaluronic acid are liver and lymph gland. Such metabolism occurs as a result of incorporation of hyaluronic acid into cells via cell membrane-localized receptors that specifically bind to hyaluronic acid, which include CD44, RHAMM, and HARE as typical examples, and the subsequent decomposition by hyaluronidase. It has been reported that the main recognition sites of both of these molecules are contiguous free carboxy groups (hexasaccharide) of hyaluronic acid (refer to Non-Patent Document 4).
Accordingly, in order to overcome the problem of hyaluronic acid such as a short residence time in blood, an attempt has been made to use as a drug carrier a modified hyaluronic acid obtained by introducing a substituent into hyaluronic acid (refer to Patent Documents 4, 5, and 6). In general, when a substituent is introduced into hyaluronic acid, the residence time in blood is elongated, and it is considered that the degree of such elongation correlates to the introduction ratio of a substituent. A modified hyaluronic acid obtained by introducing substituents into various sites of hyaluronic acid has been reported. Among others, introduction of a substituent into a carboxy group in the glucuronic acid portion of hyaluronic acid via an amide bond that is hardly hydrolyzed is effective for inhibiting the obtained modified hyaluronic acid to bind to a hyaluronic acid receptor. Thus, it is considered that the modified hyaluronic acid is excellent in terms of the residence time in blood.
On the other hand, it has been reported that hyaluronic acid is converted to a tetrabutylammonium salt, and that the salt is then reacted with a substituent in dimethyl sulfoxide, so as to obtain a modified hyaluronic acid wherein the carboxy group of the hyaluronic acid has been amidated (refer to Patent Document 7). However, this invention has been made to prepare a cross-linked material, and it has not been intended to elongate the residence time in blood. What is more, 1,1-carbonyldiimidazole (hereinafter also referred to as “CDI”) is used as a condensing agent in this invention. In our studies, it has been confirmed that although CDI is used as a condensing agent, it is not possible to obtain a hyaluronic acid derivative having a sufficiently elongated residence time in blood (which is sufficiently resistant to decomposition by hyaluronidase), and further that the molecular weight of hyaluronic acid is significantly decreased during the reaction.
Other than these reports, there has been reported a case where a substituent had been introduced into the carboxy group of hyaluronic acid in a mixed solvent consisting of water and a polar organic solvent (refer to Patent Document 8). However, whether or not the obtained modified hyaluronic acid brings on an elongation of the residence time in blood is not mentioned at all in this report.
Thus, a water-soluble modified hyaluronic acid that is practically used as a drug carrier, and in particular, a water-soluble modified hyaluronic acid whose residence time in blood has been extended up to a practical level, has been unknown. The production method thereof has also been unknown.
Pharmaceutical preparations of proteins or peptides having pharmacological effects, which have been practically used in recent years, generally have a short half-life period in blood. In addition, since the majority of them are injectable formulations that involve frequent administration, administration of such preparations imposes an enormous burden upon patients. Accordingly, it is desired that a practical sustained-release formulation of a protein or peptide drug, which exhibits drug effect even if it is used in an amount as small as possible, and the number of administration of which is also small, be developed. Moreover, a drug comprising a low-molecular-weight compound is highly required as a prolonged action drug because of its short half-life in blood.
For example, it has been attempted to prepare a sustained-release formulation composed of a biodegradable polymer such as polylactic acid-glycolic acid copolymer (hereinafter also referred to as “PLGA”) as a substrate. However, regarding preparation of such a formulation, denaturation and aggregation of a protein caused by the hydrophobicity of the substrate, a drying process, and a decrease in pH, has been reported (refer to Non-Patent Documents 5 and 6). A sustained-release formulation using a hydrophilic hydrogel as a substrate has been reported. However, it has not yet been in practical use. To date, a sustained-release formulation, all of the encapsulation ratio of a protein or peptide, the recovery ratio, and safety of which reach the practical level, has not been realized.
A cross-linking method using, as a sustained-release substrate, HA that has nonantigenicity, nonmutagenicity, nontoxicity and biodegradability, and that is considered to be preferable in terms of safety, and the sustained-release of a protein or peptide drug from HA gel, have been frequently reported. Known methods of gelating HA via a chemical cross-link may include the carbodiimide method (refer to Patent Document 9), the divinyl sulfone method (refer to Patent Document 10), and the glycidylether method (refer to Patent Document 11). In general, in the case that a protein or peptide is encapsulated in gel by a method of introducing a protein or peptide after cross-linking, the introduction ratio is low due to problems such as compatibility of HA with a protein or peptide, electrostatic repulsion or the like. On the other hand, when in situ cross-linking is performed in the presence of a protein or peptide, there is an advantage in that a protein or peptide is deposited at a high encapsulation ratio. There have been reports regarding a formulation, whereby a protein or peptide is encapsulated in HA gel via such in situ cross-linking and is then subjected to sustained-release (refer to Patent Document 12, for example). However, if HA is subjected to in situ cross-linking by such a method in the presence of a protein or peptide, there is a problem in terms of the recovery ratio. For example, there has been a method of cross-linking an HA derivative (hereinafter also referred to as “HA-HZ”), into which a hydrazide group (hereinafter also referred to as “HZ”) has been introduced, with a cross-linking agent consisting of an N-hydroxysuccinimide (hereinafter also referred to as “NHS”) ester (refer to Patent Document 13). Since this method is applied for the purpose of in situ cross-linking under physiological conditions, a cross-link formation reaction is carried out at pH of 7.4 to 8.5. According to the studies of the present inventors, however, the recovery ratio of a protein or peptide obtained from HA gel by this method is low. This is because a part of a protein or peptide (mainly an amino group) is reacted with a cross-linking agent during the cross-linking reaction, and thus the protein is cross-linked. In addition, a denatured protein or peptide that remains in the gel has a decreased biological activity, and thus it is rather problematic in that it becomes an antigen. The release of the encapsulated drug at a high recovery ratio is an essential condition as a pharmaceutical. A method of chemically cross-linking and gelating HA without allowing a protein or peptide to react with a cross-linking agent has not yet been known. Moreover, as a method of encapsulation of a protein or peptide at a high recovery ratio, it has been reported that an unsaturated functional group is cross-linked via a nucleophilic addition reaction using polyethylene glycol (PEG) as a substrate (refer to Patent Document 14). However, this method is problematic in that non-biodegradable PEG fragments remain. In order to solve such problems, a hyaluronic acid gel, of which the reactivity with a protein or peptide is suppressed to a minimum, has been reported (refer to Patent Document 15). However, the sustained-release period thereof is only approximately 1 week.
Furthermore, in order to produce an injectable formulation from such a drug sustained-release substance, it is necessary to convert the above substance to fine particles. A spray dryer has been broadly used for such studies, and microparticulation of insulin (refer to Non-Patent Documents 7 and 8) and an rh anti-IgE antibody (refer to Non-Patent Document 9) has been reported. Further, encapsulation of a drug in fine particles of hyaluronic acid has also been reported (refer to Patent Documents 16 and 17). However, in these cases, since such fine particles are dissolved under the skin for a short time, the drug sustained-release period thereof is extremely short, and thus the practicability for sustained-release purpose is low. Furthermore, it has also been reported that a cross-linking reaction of chitosan is carried out during spray drying, so as to encapsulate a low-molecular-weight drug (refer to Non-Patent Document 10). However, in the case of this method, since the release period is short, like a few minutes, and since aldehyde used as a cross-linking agent has a high reactivity with a functional group such as an amino group, this method cannot be applied to a protein, a peptide, and other low-molecular-weight drugs having functional groups such as an amino group.
Thus, a practical sustained-release formulation using a water-soluble modified hyaluronic acid as a substrate has been unknown, and the production method thereof has also been unknown.    Patent Document 1: International Publication WO 92/06714    Patent Document 2: Japanese Patent Application No. 2001-81103 A    Patent Document 3: Japanese Patent Application No. 2-273176 A    Patent Document 4: Japanese Patent Application No. 5-85942 A    Patent Document 5: International Publication WO 01/05434    Patent Document 6: International Publication WO 01/60412    Patent Document 7: Japanese Patent Application No. 2002-519481A    Patent Document 8: International Publication WO 94/19376    Patent Document 9: International Publication WO 94/02517    Patent Document 10: Japanese Patent Application No. 61-138601A    Patent Document 11: Japanese Patent Application No. 5-140201A    Patent Document 12: U.S. Pat. No. 5,827,937    Patent Document 13: International Publication WO 95/15168    Patent Document 14: International Publication WO 00/44808    Patent Document 15: International Publication WO 04/046200    Patent Document 16: Japanese Patent No. 3445283    Patent Document 17: International Publication WO 96/18647    Non-Patent Document 1: Int. J. Pharm., Vol. 255, pp. 167-174, 2003    Non-Patent Document 2: J. Control. Rel., Vol. 88, pp. 35-42, 2003    Non-Patent Document 3: J. Inter. Med., Vol. 242, pp. 27-33, 1997    Non-Patent Document 4: Exp. Cell Res., Vol. 228, pp. 216-228, 1996    Non-Patent Document 5: J. Pharm. Sci., Vol. 88, pp. 166-173, 1999    Non-Patent Document 6: J. Microencapsulation, Vol. 15, pp. 699-713, 1998    Non-Patent Document 7: Int. J. Pharm., Vol. 233, pp. 227-237, 2002    Non-Patent Document 8: J. Control. Rel., Vol. 91, pp. 385-394, 2003    Non-Patent Document 9: Biotech. and Bioeng., Vol. 60, pp. 301-309, 1998    Non-Patent Document 10: Int. J. Pharm., Vol. 187, pp. 53-65, 1999